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Cells Feb 2022Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing... (Review)
Review
Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4-7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2-3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.
Topics: Cell Culture Techniques; Cryopreservation; Feeder Cells; Freezing; Induced Pluripotent Stem Cells
PubMed: 35269421
DOI: 10.3390/cells11050799 -
Health Technology Assessment... May 2022Freezing all embryos, followed by thawing and transferring them into the uterine cavity at a later stage (freeze-all), instead of fresh-embryo transfer may lead to... (Randomized Controlled Trial)
Randomized Controlled Trial
BACKGROUND
Freezing all embryos, followed by thawing and transferring them into the uterine cavity at a later stage (freeze-all), instead of fresh-embryo transfer may lead to improved pregnancy rates and fewer complications during in vitro fertilisation and pregnancies resulting from it.
OBJECTIVE
We aimed to evaluate if a policy of freeze-all results in a higher healthy baby rate than the current policy of transferring fresh embryos.
DESIGN
This was a pragmatic, multicentre, two-arm, parallel-group, non-blinded, randomised controlled trial.
SETTING
Eighteen in vitro fertilisation clinics across the UK participated from February 2016 to April 2019.
PARTICIPANTS
Couples undergoing their first, second or third cycle of in vitro fertilisation treatment in which the female partner was aged < 42 years.
INTERVENTIONS
If at least three good-quality embryos were present on day 3 of embryo development, couples were randomly allocated to either freeze-all (intervention) or fresh-embryo transfer (control).
OUTCOMES
The primary outcome was a healthy baby, defined as a live, singleton baby born at term, with an appropriate weight for their gestation. Secondary outcomes included ovarian hyperstimulation, live birth and clinical pregnancy rates, complications of pregnancy and childbirth, health economic outcome, and State-Trait Anxiety Inventory scores.
RESULTS
A total of 1578 couples were consented and 619 couples were randomised. Most non-randomisations were because of the non-availability of at least three good-quality embryos ( = 476). Of the couples randomised, 117 (19%) did not adhere to the allocated intervention. The rate of non-adherence was higher in the freeze-all arm, with the leading reason being patient choice. The intention-to-treat analysis showed a healthy baby rate of 20.3% in the freeze-all arm and 24.4% in the fresh-embryo transfer arm (risk ratio 0.84, 95% confidence interval 0.62 to 1.15). Similar results were obtained using complier-average causal effect analysis (risk ratio 0.77, 95% confidence interval 0.44 to 1.10), per-protocol analysis (risk ratio 0.87, 95% confidence interval 0.59 to 1.26) and as-treated analysis (risk ratio 0.91, 95% confidence interval 0.64 to 1.29). The risk of ovarian hyperstimulation was 3.6% in the freeze-all arm and 8.1% in the fresh-embryo transfer arm (risk ratio 0.44, 99% confidence interval 0.15 to 1.30). There were no statistically significant differences between the freeze-all and the fresh-embryo transfer arms in the live birth rates (28.3% vs. 34.3%; risk ratio 0.83, 99% confidence interval 0.65 to 1.06) and clinical pregnancy rates (33.9% vs. 40.1%; risk ratio 0.85, 99% confidence interval 0.65 to 1.11). There was no statistically significant difference in anxiety scores for male participants (mean difference 0.1, 99% confidence interval -2.4 to 2.6) and female participants (mean difference 0.0, 99% confidence interval -2.2 to 2.2) between the arms. The economic analysis showed that freeze-all had a low probability of being cost-effective in terms of the incremental cost per healthy baby and incremental cost per live birth.
LIMITATIONS
We were unable to reach the original planned sample size of 1086 and the rate of non-adherence to the allocated intervention was much higher than expected.
CONCLUSION
When efficacy, safety and costs are considered, freeze-all is not better than fresh-embryo transfer.
TRIAL REGISTRATION
This trial is registered as ISRCTN61225414.
FUNDING
This project was funded by the National Institute for Health and Care Research (NIHR) Health Technology Assessment programme and will be published in full in ; Vol. 26, No. 25. See the NIHR Journals Library website for further project information.
Topics: Embryo Transfer; Female; Fertilization in Vitro; Freezing; Humans; Live Birth; Male; Ovarian Hyperstimulation Syndrome; Pregnancy; Pregnancy Rate
PubMed: 35603917
DOI: 10.3310/AEFU1104 -
Anatomical Record (Hoboken, N.J. : 2007) Jan 2022Preventing postmortem deterioration of soft-tissues is an important requisite of anatomical research. In order to provide corrections for potential myological...
Preventing postmortem deterioration of soft-tissues is an important requisite of anatomical research. In order to provide corrections for potential myological distortions, this study quantifies the acute effects of freezing, formalin fixation and ethanol storage using muscles from (n = 46) rabbits (Oryctolagus cuniculus). Bilateral dissections of specific muscles were performed and each side was assigned to a different preparation group (fresh, formalin fixation only, fixation followed by short duration ethanol storage, and freezing once or twice). We demonstrate that short-term freezing at -20C and thawing have no significant effect on muscle mass, volume, and density while short-term formalin fixation and ethanol storage significantly reduces mass and volume (density remains relatively constant.) Although freezing may have less of an effect on the gross morphometric characteristics of the musculature than ethanol storage, slow freezing damages muscle microanatomy, and therefore, faster freezing and other modes of preservation such as formalin fixation and ethanol storage may be preferable. Based on our results, we derived the following correction factors for each preparation: the mass of specimens stored in 70% ethanol should be multiplied by 1.69 to approximate fresh muscle mass, and specimens fixed in 10% formalin multiplied by 1.32. Although not significant, specimens frozen-once were slightly less massive and could be multiplied by 1.03 (frozen-twice ×1.09). The volumetric corrections are: ethanol 1.64; 10% formalin 1.32; frozen-once 1.03; frozen-twice 1.10. While the density of ethanol preserved specimens is slightly less than that of fresh ones (correction: 1.03), those preserved in formalin and frozen maintain nearly the same density.
Topics: Animals; Ethanol; Formaldehyde; Freezing; Muscles; Rabbits; Tissue Fixation
PubMed: 33843149
DOI: 10.1002/ar.24639 -
Global Change Biology Jul 2019Temperate plants are at risk of being exposed to late spring freezes. These freeze events-often called false springs-are one of the strongest factors determining... (Review)
Review
Temperate plants are at risk of being exposed to late spring freezes. These freeze events-often called false springs-are one of the strongest factors determining temperate plants species range limits and can impose high ecological and economic damage. As climate change may alter the prevalence and severity of false springs, our ability to forecast such events has become more critical, and it has led to a growing body of research. Many false spring studies largely simplify the myriad complexities involved in assessing false spring risks and damage. While these studies have helped advance the field and may provide useful estimates at large scales, studies at the individual to community levels must integrate more complexity for accurate predictions of plant damage from late spring freezes. Here, we review current metrics of false spring, and how, when, and where plants are most at risk of freeze damage. We highlight how life stage, functional group, species differences in morphology and phenology, and regional climatic differences contribute to the damage potential of false springs. More studies aimed at understanding relationships among species tolerance and avoidance strategies, climatic regimes, and the environmental cues that underlie spring phenology would improve predictions at all biological levels. An integrated approach to assessing past and future spring freeze damage would provide novel insights into fundamental plant biology and offer more robust predictions as climate change progresses, which are essential for mitigating the adverse ecological and economic effects of false springs.
Topics: Climate Change; Freezing; Plants; Seasons
PubMed: 30953573
DOI: 10.1111/gcb.14642 -
Molecules (Basel, Switzerland) Feb 2021It is counterintuitive that chemical reactions can be accelerated by freezing, but this amazing phenomenon was discovered as early as the 1960s. In frozen systems, the... (Review)
Review
It is counterintuitive that chemical reactions can be accelerated by freezing, but this amazing phenomenon was discovered as early as the 1960s. In frozen systems, the increase in reaction rate is caused by various mechanisms and the freeze concentration effect is the main reason for the observed acceleration. Some accelerated reactions have great application value in the chemistry synthesis and environmental fields; at the same time, certain reactions accelerated at low temperature during the storage of food, medicine, and biological products should cause concern. The study of reactions accelerated by freezing will overturn common sense and provide a new strategy for researchers in the chemistry field. In this review, we mainly introduce various mechanisms for accelerating reactions induced by freezing and summarize a variety of accelerated cryochemical reactions and their applications.
Topics: Animals; Freezing; Humans; Hydrogen-Ion Concentration; Kinetics; Organic Chemicals; Peptide Fragments; Polymerization; Proteins
PubMed: 33535547
DOI: 10.3390/molecules26030750 -
Genes Jul 2020The ability of yeast to survive freezing and thawing is most frequently considered in the context of cryopreservation, a practical step in both industrial and research... (Review)
Review
The ability of yeast to survive freezing and thawing is most frequently considered in the context of cryopreservation, a practical step in both industrial and research applications of these organisms. However, it also relates to an evolved ability to withstand freeze-thaw stress that is integrated with a larger network of survival responses. These responses vary between different strains and species of yeast according to the environments to which they are adapted, and the basis of this adaptation appears to be both conditioned and genetic in origin. This review article briefly touches upon common yeast cryopreservation methods and describes in detail what is known about the biochemical and genetic determinants of cell viability following freeze-thaw stress. While we focus on the budding yeast , in which the freeze-thaw stress response is best understood, we also highlight the emerging diversity of yeast freeze-thaw responses as a manifestation of biodiversity among these organisms.
Topics: Adaptation, Physiological; Cryopreservation; Freezing; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 32707778
DOI: 10.3390/genes11080835 -
Role of freezing-induced myofibrillar protein denaturation in the generation of thaw loss: A review.Meat Science Aug 2022Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from... (Review)
Review
Formation of thaw loss cannot generally be avoided when meat is frozen and then thawed. Explanations have mainly focused on the damage to muscle fibers resulting from ice crystallization and the freezing-induced denaturation of myofibrillar proteins, the latter of which has, however, not received much research focus. This review discusses the relationship between myofibrillar protein denaturation and water-holding capacity of meat in freezing-thawing with the aim to improve the understanding the relative importance of protein denaturation in the formation of thaw loss. The contribution of decreased pH and high ionic strength in the unfrozen water in freezing is emphasized and we hypothesize that these two factors are causing protein denaturation and conformational changes within muscle fibers, and consequently loss of water-holding capacity. Slow freezing produces more thaw loss than fast freezing, and this is discussed here in relation to the impacts on myofibrillar protein denaturation induced by the freezing rate.
Topics: Freezing; Meat; Protein Denaturation; Proteins; Water
PubMed: 35533633
DOI: 10.1016/j.meatsci.2022.108841 -
The ISME Journal Apr 2021Climate change alters frequencies and intensities of soil drying-rewetting and freezing-thawing cycles. These fluctuations affect soil water availability, a crucial...
Climate change alters frequencies and intensities of soil drying-rewetting and freezing-thawing cycles. These fluctuations affect soil water availability, a crucial driver of soil microbial activity. While these fluctuations are leaving imprints on soil microbiome structures, the question remains if the legacy of one type of weather fluctuation (e.g., drying-rewetting) affects the community response to the other (e.g., freezing-thawing). As both phenomenons give similar water availability fluctuations, we hypothesized that freezing-thawing and drying-rewetting cycles have similar effects on the soil microbiome. We tested this hypothesis by establishing targeted microcosm experiments. We created a legacy by exposing soil samples to a freezing-thawing or drying-rewetting cycle (phase 1), followed by an additional drying-rewetting or freezing-thawing cycle (phase 2). We measured soil respiration and analyzed soil microbiome structures. Across experiments, larger CO pulses and changes in microbiome structures were observed after rewetting than thawing. Drying-rewetting legacy affected the microbiome and CO emissions upon the following freezing-thawing cycle. Conversely, freezing-thawing legacy did not affect the microbial response to the drying-rewetting cycle. Our results suggest that drying-rewetting cycles have stronger effects on soil microbial communities and CO production than freezing-thawing cycles and that this pattern is mediated by sustained changes in soil microbiome structures.
Topics: Climate Change; Desiccation; Freezing; Soil; Soil Microbiology
PubMed: 33408369
DOI: 10.1038/s41396-020-00844-3 -
Faraday Discussions Nov 2022Grid freezing is a critical step for successful cryo-transmission electron microscopy, and optimising freezing conditions is a considerable bottleneck in many projects....
Grid freezing is a critical step for successful cryo-transmission electron microscopy, and optimising freezing conditions is a considerable bottleneck in many projects. To improve reproducibility in grid preparation, temperature- and humidity-controlled chambers were built into the second generation of plunge-freezers, including the ThermoFisherScientific Vitrobot and Leica GP. Since then, for most published structures, the proteins were plunge-frozen from a cold, humid environment. This provides two benefits: many proteins are more stable at 4 °C than room temperature, and both the low temperature and the humidity help control evaporation of the tiny drop of liquid. However, for optimal stability, certain samples may have different requirements. Here, we describe various (reversible) adaptations made to a Leica GP2 system to accommodate several samples with special handling requirements: a protein that is sensitive to both light and oxygen, a sample that needs to be kept at 37 °C throughout the plunge-freezing process, and a method to freeze a polymer that gels at 37 °C in its gelled state. While some of these methods are specific to these specimens, we hope sharing the ideas behind them will help people who are dealing with tricky protein samples.
Topics: Humans; Freezing; Workflow; Reproducibility of Results; Cryoelectron Microscopy
PubMed: 35913403
DOI: 10.1039/d2fd00060a -
Planta May 2021Anatomical, metabolic and microbial factors were identified that contribute to sequential freezing in wheat leaves and likely contribute to supercooling in the youngest...
Anatomical, metabolic and microbial factors were identified that contribute to sequential freezing in wheat leaves and likely contribute to supercooling in the youngest leaves and potentially meristematic regions. Infrared thermography (IR) has been used to observe wheat leaves freezing independently and in an age-related sequence with older leaves freezing first. To determine mechanisms that might explain this sequence of freezing several analytical approaches were used: (1) The size of xylem vessels, in proximity to where freezing initiated, was measured to see if capillary freezing point depression explained sequential freezing. The sequence of freezing in the four youngest leaves was correlated, with the largest vessels freezing first. (2) Carbohydrate and amino acids were analyzed to determine if solute concentrations as well as interactions with membranes explained the freezing sequence. Sucrose was highly correlated to the freezing sequence for all leaves suggesting a prominent role for this sugar as compared to other simple sugars and fructans. Among individual free amino acids proline and serine were correlated to the freezing sequence, with younger leaves having the highest concentrations. (3) Microflora within and on leaf surfaces were determined to measure potential freezing initiation. Levels of bacteria and fungi were correlated to the freezing sequence for all leaves, and species or genera associated with high ice nucleation activity were absent in younger leaves. Moisture content and transcript expression of ice binding proteins were also measured. As expected, our results show that no single mechanism explains the freezing sequence observed via infrared analyses. While these multiple mechanisms are operative at different levels according to the leaf age, they seem to converge when it comes to the protection of vital meristematic tissues. This provides potential phenotypic characters that could be used by breeders to develop more winter-hardy genotypes.
Topics: Freezing; Ice; Plant Leaves; Triticum; Xylem
PubMed: 34014374
DOI: 10.1007/s00425-021-03637-w